Hans L. Hoogveld

The Timescale of Phenotypic Plasticity and Its Impact on Competition in Fluctuating Environments

Although phenotypic plasticity can be advantageous in fluctuating environments, it may come too late if the environment changes fast. Complementary chromatic adaptation is a colorful form of phenotypic plasticity, where cyanobacteria tune their pigmentation to the prevailing light spectrum. Here, we study the timescale of chromatic adaptation and its impact on competition among phytoplankton species exposed to fluctuating light colors. We parameterized a resource competition model using monoculture experiments with green and red picocyanobacteria and the cyanobacterium Pseudanabaena, which can change its color within ∼7 days by chromatic adaptation. The model predictions were tested in competition experiments, where the incident light color switched between red and green at different frequencies (slow, intermediate, and fast). Pseudanabaena (the flexible phenotype) competitively excluded the green and red picocyanobacteria in all competition experiments. Strikingly, the rate of competitive exclusion was much faster when the flexible phenotype had sufficient time to fully adjust its pigmentation. Thus, the flexible phenotype benefited from its phenotypic plasticity if fluctuations in light color were relatively slow, corresponding to slow mixing processes or infrequent storms in their natural habitat. This shows that the timescale of phenotypic plasticity plays a key role during species interactions in fluctuating environments.

Detecting the phosphate status of phytoplankton by enzyme-labelled fluorescence and flow cytometry

The novel phosphatase substrate, ELF-97 phosphate, yields intensely green fluorescent precipitates of ELF-97 alcohol (ELFA) upon enzymatic dephosphorylation, and thereby traces phosphatase activity back to its producer. In this study, we show that ELFA fluorescence is a useful tool in flow cytometric analysis of natural phytoplankton populations. Presence of endogenous fluorescent pigments allowed flow cytometric distinction of clusters in the phytoplankton community in Lake Loosdrecht (The Netherlands): Eukaryotes (diatoms and green algae), chlorophyll a and b containing but phycobilin-less cyanobacteria (Prochlorothrix hollandica), and phycocyanin-containing cyanobacteria (predominantly Limnothrix sp.). Several, but not all tested cyanobacteria showed ELFA fluorescence. The dominant Limnothrix sp. possesses a derepressible phosphatase, whereas the second most abundant strain, P. hollandica, did not have phosphatase activity. Within both natural and cultured populations of Limnothrix sp. we found discernible levels of ELFA fluorescence, indicating the presence of subpopulations with different physiological characteristics.

LINKING FLOW CYTOMETRIC CELL SORTING AND COMPOUND‐SPECIFIC 13C‐ANALYSIS TO DETERMINE POPULATION‐SPECIFIC ISOTOPIC SIGNATURES AND GROWTH RATES IN CYANOBACTERIA‐DOMINATED LAKE PLANKTON1

A novel methodology was applied to determine the δ13C signatures of natural cyanobacterial and algal populations by combined compound‐specific isotope ratio mass spectrometry and pyrolytic methylation‐gas chromatography (Py‐GC‐IRMS) of the fatty acids released from phytoplankton fractions collected using fluorescence‐activated cell sorting. Py‐GC‐IRMS provided direct analysis of the very small samples (<200 ng total C) derived from the cell sorting of individual phototrophic populations, while minimizing the chances on contamination and loss in sample handling. Despite trichome lengths exceeding the diameter of the sort droplets, filamentous cyanobacteria were amenable to population‐specific cell sorting. In concert with 13C‐CO2 labeling, the combined use of flow cytometric cell sorting and Py‐GC‐IRMS enabled both the assessment of standing stocks and of population‐specific growth rates of the predominant cyanobacterial and algal taxa in Lake Loosdrecht (The Netherlands). Filamentous prochlorophytes, formerly the dominant cyanobacterial taxon in the lake, appeared less abundant in recent years and exhibited growth rates 30%–40% lower than the rates recorded for oscillatorioid populations. Diatom and green algal populations grew at rates 4‐ to 10‐fold higher than filamentous cyanobacteria and are thus important for the lakes carbon budget. This approach offers new possibilities in studying plankton dynamics at a resolution not feasible in the past.

Nitrite as a Stimulus for Ammonia-Starved Nitrosomonas europaea

ABSTRACT Ammonia-starved cells of Nitrosomonas europaea are able to preserve a high level of ammonia-oxidizing activity in the absence of ammonium. However, when the nitrite-oxidizing cells that form part of the natural nitrifying community do not keep pace with the ammonia-oxidizing cells, nitrite accumulates and may subsequently inhibit ammonia oxidation. The maintenance of a high ammonia-oxidizing capacity during starvation is then nullified. In this study we demonstrated that cells of N. europaea starved for ammonia were not sensitive to nitrite, either when they were starved in the presence of nitrite or when nitrite was supplied simultaneously with fresh ammonium. In the latter case, the initial ammonia-oxidizing activity of starved cells was stimulated at least fivefold.

Observations on cyanobacterial population collapse in eutrophic lake water.

In two laboratory-scale enclosures of water from the shallow, eutrophic Lake Loosdrecht (the Netherlands), the predominating filamentous cyanobacteria grew vigorously for 2 weeks, but then their populations simultaneously collapsed, whereas coccoid cyanobacteria and algae persisted . The collapse coincided with a short peak in the counts of virus-like particles. Transmission electron microscopy showed the morphotype Myoviridae phages, with isometric heads of about 90 nm outer diameter and >100-nm long tails, that occurred free, attached to and emerging from cyanobacterial cells. Also observed were other virus-like particles of various morphology. Similar mass mortality of the filamentous cyanobacteria occurred in later experiments, but not in Lake Loosdrecht. As applies to lakes in general, this lake exhibits high abundance of virus-like particles. The share and dynamics of infectious cyanophages remain to be established, and it is as yet unknown which factors primarily stabilize the host–cyanophage relationship. Observations on shallow, eutrophic lakes elsewhere indicate that the cyanophage control may also fail in natural water bodies exhibiting predominance of filamentous cyanobacteria. Rapid supply of nutrients appeared to be a common history of mass mortality of cyanobacteria and algae in laboratory and outdoor enclosures as well as in highly eutrophic lakes.

Dynamic modelling of viral impact on cyanobacterial populations in shallow lakes: Implications of burst size

Laboratory experiments with whole water-columns from shallow, eutrophic lakes repeatedly showed collapse of the predominant filamentous cyanobacteria. The collapse could be due to viral activity, from the evidence of electron microscopy of infected cyanobacterial cells and observed dynamics of virus-like particles. Burst-size effects on single-host single-virus dynamics was modelled for nutrient-replete growth of the cyanobacteria and fixed viral decay rate in the water column. The model combined previously published equations for nutrient-replete cyanobacterial growth and virus–host relationship. According to the model results, burst sizes greater than 200 to 400 virions per cell would result in host extinction, whereas lower numbers would allow coexistence, and even stable population densities of host and virus. High-nutrient status of the host cells might accommodate a large burst size. The ecological implication could be that burst-size increase accompanying a transition from phosphorus to light-limited cyanobacterial growth might destabilize the virus–host interaction and result in the population collapse observed in the experiments.

Optimizing the setup of a flow cytometric cell sorter for efficient quantitative sorting of long filamentous cyanobacteria

Heterogeneity within natural phytoplankton communities makes it very difficult to analyze parameters at the single‐cell level. Flow cytometric sorting is therefore a useful tool in aquatic sciences, as it provides material for post‐sort analysis and culturing. Sorting subpopulations from natural communities, however, often requires handling morphologically diverse and complex particles with various abundances. Long particles, such as filament‐forming cyanobacteria (>100‐μm long), prove very difficult to handle. These potentially toxic organisms are widespread in eutrophic systems and have important ecological consequences. Being able to sort filamentous cyanobacteria efficiently and as viable cells is therefore highly desirable when studying factors associated with their toxicity and occurrence. This unconventional sorting requires extensive user experience and special instrument setup. We have investigated the effect of hydrodynamic and electromechanical components of a flow cytometer, and sorting protocol on the quantitative sorting efficiency of these long particles using two filamentous cyanobacterial strains with average lengths of ∼100 and ∼300 μm. Sorting efficiency ranged from 9.4 to 96.0% and was significantly affected by filament length, sorting envelope, drop delay (dd), and for the long species also by tip size, but not by cycle time. Filaments survived sorting and were not damaged. The optimal settings found for the modular MoFlo® cell‐sorter to sort the filaments were a 100‐μm flow tip at 30 psi (207 kPa) with a three‐droplet envelope in Enrich mode while using an extended analysis time of 17.6 μs and an intermediate plate charge and deflection percentage combination of 3,000 V/60%, combined with a dd 0 for the cultures with 100‐μm filaments and dd +1 for the culture with 300‐μm filaments. To the best of our knowledge, the filaments up to 1063.5 μm sorted in this study are the longest ever sorted.

Phylogenetic Characterization of Phosphatase-Expressing Bacterial Communities in Baltic Sea Sediments

Phosphate release from sediments hampers the remediation of aquatic systems from a eutrophic state. Microbial phosphatases in sediments release phosphorus during organic matter degradation. Despite the important role of phosphatase-expressing bacteria, the identity of these bacteria in sediments is largely unknown. We herein presented a culture-independent method to phylogenetically characterize phosphatase-expressing bacteria in sediments. We labeled whole-cell extracts of Baltic Sea sediments with an artificial phosphatase substrate and sorted phosphatase-expressing cells with a flow cytometer. Their phylogenetic affiliation was determined by Denaturing Gradient Gel Electrophoresis. The phosphatase-expressing bacterial community coarsely reflected the whole-cell bacterial community, with a similar dominance of Alphaproteobacteria.

Erratum: Optimizing the setup of a flow cytometric cell sorter for efficient quantitative sorting of long filaments

This article corrects: Optimizing the setup of a flow cytometric cell sorter for efficient quantitative sorting of long filamentous cyanobacteria Vol. 77A, Issue 10, 911–924